Conditions in which cartilage calcification is aberrant affect both the new born and the elderly (e.g., birth defects, growth deformities, rickets, and unwanted cartilage calcification in end-stage osteoarthritis and "tissue engineered" cartilage repair. To understand and manage these conditions it is essential to under- stand physiologic cartilage calcification. Based on results of the previous funding period we plan to test the hypotheses (1) that cartilage calcification does not require prior apoptosis in the mouse, as we have already demonstrated in the chick, (2) that there is a family of extracellular phosphorylated matrix proteins that are important for regulation of mineralization in the growth plate, (3) that.aggrecan degradation is required for physiologic cartilage calcification to occur, and (4) that there are certain key regulators and matrix factors that differentiate the culture that will be mineralizable from non-mineralizable cultures. To test these hypotheses and gain additional insights into the mechanism of chondrocyte-mediated calcification we propose to use a mouse cell line along with the chick limb-bud mesenchymal cells to allow access to additional crucial reagents. With the recent availability of the chick gene array we are able to look for the "key factors" that differentiate mineralizing and non-mineralizing cultures in the chick and confirm them in the mouse. Our primary outcome for all these studies will be the amount and properties of the mineral that is deposited. The specific aims are: 1: To verify that chondrocytes apoptosis is not essential for cartilage calcification in the mouse cell line known to under go endochondral ossification. 2: To expand our ongoing studies of the importance of modification of matrix proteins by specific matrix metalloproteases (MMPs) and cathepsins. 3: To determine which phosphoproteins made by hypertrophic chondrocytes and found in calcified cartilage are responsible for the initiation and regulation of cartilage calcification using a combination of immuno-localization, immuno-blocking, and addition of extraneous protein to cultures, and to verify any critical role with knockdown experiments using RNAi. 4: To use the chick gene array to discover novel factors that distinguish mineralizing from non-mineralizing micromass cultures, and verify these in both the chick and the mouse cell lines by immuno-localization, immuno-blocking, over expressing or blocking (RNAi) the expression of specific genes, and demonstrating their effects on mineralization.